Our knowledge on the molecular basis of pluripotency and differentiation remains limited. This is mostly due to the fact that molecular and genetic tools developed in model systems are mostly lacking in human embryonic stem cells (hESCs). To overcome this shortcoming, I propose to use a transposon mediated genetic screen directly in hESCs to identify mutants and isolate genes that control the ability of human cells to differentiate. To perform our screen we will use a genetically marked hESC line, RUES2 (originally derived and characterized in my laboratory and part of the NIH registry), that has a single copy of two types of insertions: (i) a stable lentiviral insertion where the human Oct4 promoter drives the expression of EGFP and Neomycin (Neo), (ii) a mutagenic transposable element equipped with a transcriptional amplifier cassette and Hygromycin (Hyg). We have previously shown this transposable element can be mobilized to hop and randomly insert into the human genome. We propose to take advantage of this property to generate mutations and perform an unbiased forward genetic screen, as described in model organisms. Using a very simple screening strategy, based on positive (EGFP) and negative selection (Neo and Hyg), 107 hESCs will be mutagenized and challenged for their ability to retain pluripotency and lose their ability to differentiate. The transposon has properties that allow the characterization of th mutated gene with a straightforward plasmid rescue strategy. Additionally, it can be excised from the human genome without leaving a footprint to generate revertants. Those, in addition to phenocopies of the mutant by independent means, will confirm the link between the gene and the mutant phenotype. This large-scale genetic screen will lead to the isolation of genes that control a cell fate decision in hESCs. As hESCs provide a window to early human embryogenesis, this knowledge will move us a step closer to the understanding of our own development. More importantly, the successful accomplishment of this project will open the door to a more systematic use of unbiased genetic screens in human cells. In turn, this will have a strong impact on both basic research and clinical applications.